Bone marrow (BM) transplantation has been used extensive to treat human malignancies (alone or as an adjunct to chemo-radio-therapies) and to correct genetic defects. Recent studies have shown that the CD34 antigen is expressed by 1-4% of human bone marrow mononuclear cells and the CD34+ CD33- cells include virtually all hematopoietic progenitor cells detected by in vitro assays. These purified progenitor cells can be highly proliferated/expanded by in vitro culture. The increasing use of purified/expanded progenitor cells (e.g., studies of hematopoiesis, immunology, auto-and allotransplantation, and gene therapy transplantation) highlights a great concern that cryopreservation and banking of purified and/or expanded progenitor cells is necessary, and a high and stable cell cryosurvival rate is required. Although cryopreservation of whole human marrow has a history of over 30 years, which is good enough to provide the basis for autologous bone marrow transplantation, there are still several reasons to believe that further investigation is necessary to establish a stable and standard cryopreservation procedure to ensure a high survival rate of purified progenitor cells from other sources, such as umbilical cord blood. Primary among these are: (a) reported cryopreservation techniques have been developed empirically and have resulted in a variation of survival rate (between 40% to over 90%); (b) the mechanism for such variation is unclear; (c) Cryopreservation procedures designed for BM cells failed to successfully preserve purified progenitor cells; (d) cell clumping and loss occur during the removal of cryoprotectant from cryopreserved cells; and (e) the quantity of CD34+ cells is limited, and the proportional injury in such a small cell population may result in a non-viable transplant. The purpose of this proposed research is to study the separate responses of the purified CD34+ human umbilical cord derived hematopoietic stem cells to underlying fundamental cryobiological factors that determine survival after freezing and thawing, and to use this information to develop procedures for preservation of these cells with high and consistent survival. Freezing and thawing subjects cells to highly abnormal osmotic conditions, how the cells respond to the environment depends on their permeability to water and cryoprotective agents (CPAs). In this proposal, a series of experiments is designed first to determine osmotic behavior of the stem cells, their permeability to water and to CPAs, and the temperature co-efficients of these permeabilities. This information will be used to select an optimal type and concentration of CPAs and to predict the optimal rate at which the cells can be cooled without lethal intracellular freezing. Finally, the interactions between cooling rate, warming rate, CPA type and concentration on cell cryosurvival will be determined. We expect that optimal cryopreservation procedures for umbilical cord derived stem cells will be different from those current used for bone marrow derived stem cells.